Loop mediated isothermal amplification assay for detection of Trichomonas vaginalis in vaginal swabs among symptomatic women from North India.

Trichomonas vaginalis is one of the most common curable sexually transmitted pathogens infecting both men and women worldwide. Unlike traditional methods such as microscopy and culture, nucleic acid amplification tests rapidly detect this agent, assisting in treatment. Conventional polymerase chain reaction (PCR), the loop-mediated isothermal amplification (LAMP), and the Xpert TV assay were evaluated using 28 microscopy positive T. vaginalis samples and 125 microscopy negative samples from symptomatic females of reproductive age. The sensitivity of all tests was 100% and the specificity was 100%, 100%, and 99·2% for PCR, Xpert TV, and LAMP, respectively. The inter-rater reliability was excellent for PCR: Xpert TV (kappa-coefficient = 1) and good for LAMP assay: Xpert TV/PCR (kappa-coefficient = 0·98) and conventional PCR: LAMP (kappa-coefficient = 0·98). The study highlights the importance of PCR for screening T. vaginalis in women, particularly in laboratories where the Xpert-TV assay is not available or not affordable. The LAMP assay showed a lower positive predictive value which merits further evaluation. SIGNIFICANCE AND IMPACT OF THE STUDY: Trichomonas vaginalis is a common sexually transmitted pathogen associated with considerable morbidity and risk of complications. Due to the limitations of traditional diagnostic modalities, three molecular assays were compared: conventional polymerase chain reaction (PCR), Xpert TV assay, and loop mediated isothermal amplification (LAMP) assay for detecting T. vaginalis in symptomatic females. All tests had a sensitivity of 100% and the inter-rater reliability was excellent for PCR: Xpert TV, and good for LAMP assay: Xpert TV/PCR. The translational impact of this study lies in the possible use of conventional PCR and LAMP in laboratories where the Xpert TV assay is not available or not affordable.

Multiplex PCR in diagnosis of M. tuberculosis and M. avium co-infection from lymph node in an AIDS patient.

A 35-year-old, HIV-seropositive male (CD4 count 41 cells/mm3) on highly active antiretroviral ( HAART) presented with fever and weight loss for 3 months and new skin lesions. He was earlier diagnosed of TB and was on anti-tubercular therapy (ATT). The retroperitoneal lymph node aspirate showed acid-fast bacilli and epithelioid cell granulomas; however, cultures remained sterile. A dual infection with Mycobacterium tuberculosis and Mycobacterium avium was diagnosed with multiplex polymerase chain reaction (MPCR). Clarithromycin was added to ATT, and on follow-up at 1 and 3 months, the patient responded well. Molecular methods like MPCR should be exploited for routine diagnosis of high-risk patients.

Mandatory public reporting of healthcare-associated infections in developed countries: how can developing countries follow?

The threat posed by increased transmission of drug-resistant pathogens within healthcare settings and from healthcare settings to the community is very real and alarming. Although the developed world has taken strong steps to curb this menace, there has been little pressure on developing countries to take any corrective action. If the reporting of alarming rates of healthcare-associated infections (HCAIs) from hospitals in India and many other developing countries was made mandatory, it would help to force stakeholders (e.g. healthcare workers, legislators, administrators and policy makers in hospitals) to acknowledge and tackle the problem. This would introduce quality control in a long neglected area of health care, and enable patient empowerment which is practically non-existent in India. Healthcare institutions should commit towards enforcing 'zero tolerance' towards lapses in prevention of HCAIs. Public pressure would force the Indian Government to acknowledge the problem, and to allocate more funds to improve resources and infrastructure; this could substantially elevate the standard of health care given to the average Indian. Despite the numerous challenges, overall public benchmarking of HCAIs is a commendable goal that would go a long way towards tackling this menace in developing countries such as India.

Molecular characterisation of drug resistance in Mycobacterium tuberculosis isolates from North India.

SETTING: A tertiary care hospital in North India. OBJECTIVES: To analyse the frequency of mutations associated with resistance to rifampicin (RMP), isoniazid (INH) and streptomycin (SM) in Mycobacterium tuberculosis. DESIGN: M. tuberculosis isolates from 171 pulmonary tuberculosis (TB) patients (newly diagnosed 102, 59.6%; retreated 69, 40.3%) were analysed. Drug susceptibility testing was performed using the proportion method and resistant isolates were characterised using the polymerase chain reaction, followed by restriction fragment length polymorphism and/or DNA sequencing, to screen for mutations in rpoB, katG, mabA-inhA and rpsL. RESULTS: Of the 171 isolates, 16.9% (29/171) were multidrug-resistant. Of the 102 newly diagnosed and 69 retreated cases, respectively 5.9% (6/102) and 33.3% (23/69) were multidrug-resistant. rpoB mutations were found in 100% (31/31) of the RMP-resistant isolates, the most common being S531L in 74.2% (23/31); katG315 mutations were found in 79.6% (35/44) of the INH-resistant isolates; however, no mabA-inhA (-15) mutation was found; rpsL mutations were found in 48.9% (24/49) of the SM-resistant isolates, and codon 43 mutation were found in 42.5% (21/49). CONCLUSIONS: This study characterises drug resistance-associated mutations in M. tuberculosis, information that could be used for the rapid screening of drug-resistant TB.

Rapid detection of rifampicin, isoniazid and streptomycin resistance in Mycobacterium tuberculosis clinical isolates by high-resolution melting curve analysis.

AIMS: This study was carried out to evaluate high-resolution melting (HRM) curve analysis assay for detection of mutations in three drug resistance-associated genes of Mycobacterium tuberculosis. METHODS AND RESULTS: Clinical isolates of Myco. tuberculosis phenotypically resistant to rifampicin (n = 29), isoniazid (n = 35) and streptomycin (n = 34) were analysed for mutations in rpoB, katG and rpsL genes, respectively, by HRM curve analysis and DNA sequencing. HRM curve assay resulted in 11 clearly distinguishable melt curves denoting eight types of mutations responsible for drug resistance. For the three drugs, respectively, the sensitivity of HRM curve assay was found to be 93·1, 80 and 61·8% compared to the phenotypic resistance patterns, and 93·1, 93·3 and 100% in comparison with the DNA sequencing. CONCLUSIONS: The sensitivity and specificity of HRM curve assay was found to be comparable to DNA sequencing. The assay offers the advantage of high throughput, single step, rapid work flow and cost effectiveness and can be utilized as a rapid screening method for detection of drug-resistant tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: HRM curve assay may prove to be an important tool for the development of rapid molecular diagnostic assays for detection of mutation-based drug resistance.